Cytoprotective biocompatible containment systems for biologically active materials and methods of making same

ABSTRACT

In accordance with the invention, there are provided methods, capsules, and delivery systems useful in preparing biological containment systems with properties (e.g., mechanical strength, capsule permeability and porosity, desired controlled release rates of the biologic or components secreted by the biologic, and immunoreactivity) that can be varied to adapt to a broader range of physiological conditions than known biological containment systems. There are also provided methods of making capsules containing cell aggregates therein, as well as the capsules formed thereby, which are useful as a quantitatively plentiful and low cost alternative to usage of freshly harvested cell aggregates (e.g., islets from pancreas), since the latter are usually available only in limited numbers.

FIELD OF THE INVENTION

[0001] The present invention relates to new forms of biocompatiblecontainment systems that envelop encapsulated or free cells or otherbiologically active materials. In a particular aspect, the presentinvention relates to a system that provides an immune barrier for thecells or other biologically active materials. In another aspect, thepresent invention relates to a system that provides enhanced migrationand aggregation of the cells or other biologically active materialswithin the containment system. In a further aspect, the presentinvention relates to a system that provides enhanced transfer of thesecretions of cells or other biologically active materials out of thecontainment system.

BACKGROUND OF THE INVENTION

[0002] Microencapsulation of cells (e.g., pancreatic islets) by analginate-PLL-alginate membrane (i.e., an alginate-poly-L-lysine-alginatemembrane) is a potential method for prevention of rejection of foreigncells by the host's immune system. By this technique, researchers areable to encapsulate living islets in a protective membrane that allowsinsulin to be secreted, yet prevents antibodies from reaching theislets, causing rejection of the cells. This membrane (or microcapsule)protects the islet from rejection and allows insulin to be secretedthrough its “pores” to maintain the diabetic in normal glucose control.

[0003] Successful transplants of microencapsulated cells have not beenclinically feasible to date due to fundamental problems of transplantrejection and/or fibrotic reaction to the microcapsule. In the treatmentof diabetes, Lim and Sun, 1980; Science 210:908, reported the firstsuccessful implantation of microencapsulated islets and describednormalization of blood sugar in diabetic rats.

[0004] However, for microencapsulated cells to be clinically useful andapplicable in humans, it is important that the capsule be biocompatible,allow adequate difflusion for the encapsulated cells to respondappropriately to a stimulatory signal and to provide the encapsulatedcells with necessary nutrients, and optionally be retrievable.Retrievability is desirable for a variety of reasons, e.g., so thataccumulation of the implanted materials can be avoided, so thatencapsulated cells can be removed from the recipient when no longerneeded or desired (e.g., when the product(s) of the encapsulated cellsare no longer needed, if the encapsulated cells fail to perform asdesired, etc.), so that encapsulated cells can be removed if/when theybecome non-viable, and the like.

[0005] Biocompatibility of encapsulated islets remains a fundamentalproblem. The term “biocompatible” is used herein in its broad sense, andrelates to the ability of the material to result in long-term in vivofunction of transplanted biological material, as well as its ability toavoid a foreign body, fibrotic response. A major problem withmicroencapsulation technology has been the occurrence of fibrousovergrowth of the epicapsular surface, resulting in cell death and earlygraft failure. Despite extensive studies, the pathological basis of thisphenomenon in alginate based capsules remains poorly understood.However, several factors have recently been identified as being involvedin graft failure, e.g., the guluronic acid/mannuronic acid content ofthe alginate employed, imperfections in the microcapsule membrane(allowing exposure of poly-L-lysine to the in vivo environment), failureof the microcapsule membrane to completely cover the cells beingencapsulated (thereby allowing exposure of the cells to the in vivoenvironment), and the like.

[0006] Accordingly, there is a need in the art for new and bettercapsules for the encapsulation of biologically active materials. Inaddition, there is a need for new methods of making capsules thatencapsulate biologically active materials while permitting variation ofcertain properties (e.g., mechanical strength, capsule permeability andporosity, desired controlled release rates of the biologic or componentssecreted by the biologic, and immunoreactivity) across broad performanceranges to address variable physiological conditions. Further, there isa, need for new methods of facilitating formation of and deliverysystems for cell aggregates.

BRIEF DESCRIPTION OF THE INVENTION

[0007] In accordance with the present invention, capsules (e.g.,microcapsules and macrocapsules) have been developed for theencapsulation of biologically active materials therein. Inventioncapsules comprise at least one biocompatible gellable material, whereinat least the outer layer of the capsule is covalently crosslinked andoptionally polyionically crosslinked (or, in the case of macrocapsulescomprising rnicrocapsules therein, either polyionically crosslinked,covalently crosslinked, or both polyionically crosslinked and covalentlycrosslinked), but not ionically crosslinked. Surprisingly, inventioncapsules permit enhanced migration and aggregation of the biologicallyactive material within the capsule and enhanced control over the releaserates of the biologically active material or components secreted by thebiologically active material, while decreasing the risk ofbiomineralization due to ions required for ionic crosslinking andenabling the biologically active material contained within the capsuleto retain a significant proportion of the functionality of theunencapsulated biologically active material.

[0008] In a further aspect of the present invention, there also havebeen developed methods of making invention capsules. One of theinvention methods comprises subjecting a capsule whose outer layer isionically crosslinked and covalently crosslinked, and optionallypolyionically crosslinked (or, in the case of macrocapsules comprisingmicrocapsules therein, ionically crosslinked and either polyionicallycrosslinked, covalently crosslinked, or both polyionically crosslinkedand covalently crosslinked), to conditions sufficient to disrupt ioniccrosslinking in at least the outer layer thereof. Surprisingly,invention methods facilitate the relatively rapid formation of inventioncapsules under conditions which are not cytotoxic, while decreasing therisk of biomineralization caused by the presence of ions required forionic crosslinking and enabling the biologically active materialcontained within the capsule to retain a significant proportion of thefunctionality of the unencapsulated biologically active material.

[0009] Additional methods of making invention capsules comprisesimultaneously subjecting a droplet comprising a suspension ofbiologically active materials in a covalently crosslinkable carrier toconditions sufficient to prevent substantial dissociation thereof andsubjecting the droplet to conditions sufficient to induce substantialcovalent crosslinking thereof. Surprisingly, these invention methodsfacilitate the relatively rapid formation of invention capsules underconditions which are not cytotoxic, while reducing to substantially zerothe risk of biomineralization caused by the presence of ions requiredfor ionic crosslinking and while enabling the biologically activematerial contained within the capsule to retain a significant proportionof the functionality of the unencapsulated biologically active material.

[0010] In a further aspect of the present invention, there also havebeen developed capsules containing cell aggregates therein, and methodsfor the production thereof. Invention capsules comprise a biocompatiblegellable material, and have a core which is not ionically crosslinked,and at least an outer layer thereof which is covalently crosslinked,polyionically crosslinked, or both covalently crosslinked andpolyionically crosslinked. Surprisingly, invention capsules permitenhanced migration and aggregation of the cell aggregates andconstituent cells within the capsule and enhanced control over therelease rates of the components secreted by the cell aggregates, whiledecreasing the risk of biomineralization due to ions required for ioniccrosslinking and enabling the cell aggregates contained within thecapsule to retain a significant proportion of the functionality of theunencapsulated cell aggregates.

[0011] Invention methods of making capsules containing cell aggregatestherein described herein comprise subjecting a capsule, comprising abiocompatible gellable material and having a core which is ionicallycrosslinked, to conditions sufficient to disrupt ionic crosslinkingtherein. Surprisingly, invention methods facilitate enhanced migrationand aggregation of the cell aggregates and their constituent componentswithin the capsule and enhanced control over the release rates of thebiologically active material or of the components secreted by thebiologically active material, while decreasing the risk ofbiomineralization due to ions required for ionic crosslinking andfacilitating the ability of the cell aggregates contained within thecapsule to retain a significant proportion of the functionality of theunencapsulated cell aggregates.

[0012] In an additional aspect of the present invention, there also havebeen developed delivery systems comprising the invention capsules.Surprisingly, invention delivery systems permit enhanced diffusion,across and throughout the capsule, of the biologically active materialcontained therein, or of the compound secreted by the biologicallyactive material contained therein, or of the compound to be catalyzedand/or reacted by the biologically active material contained therein,while decreasing the risk of biomineralization due to ions required forionic crosslinking and enabling the biologically active materialcontained within the capsule to retain a significant proportion of thefunctionality of the unencapsulated biologically active material.

[0013] The present invention provides many advantages over the art. Forexample, invention methods, capsules, and delivery systems are useful inpreparing biological containment systems with properties (e.g.,mechanical strength, capsule permeability and porosity, desiredcontrolled release rates of the biologic or components secreted by thebiologic, and immunoreactivity) that can be varied to adapt to a broaderrange of physiological conditions. This variation and adaptability aredue to the broader range of ratios of ionic to covalent linkages in thebiocompatible gellable material permitted by the present invention.Further, invention methods of making capsules containing cell aggregatestherein, as well as the capsules formed thereby, are useful as aquantitatively plentiful and low cost alternative to usage of freshlyharvested cell aggregates (e.g., islets from pancreas), since the latterare usually available only in limited numbers. Other advantages of thepresent invention can be readily recognized by those of ordinary skillin the art upon inspection of the detailed description and appendedclaims provided herewith.

DETAILED DESCRIPTION OF THE INVENTION

[0014] In accordance with the present invention, there are providedmicrocapsules containing biologically active materials therein.Invention microcapsules comprise an ionically crosslinkablebiocompatible gellable material, wherein at least the outer layer ofsaid microcapsule is covalently crosslinked and optionally polyionicallycrosslinked, but not ionically crosslinked.

[0015] As utilized herein, the term “microcapsule” includes capsules ofbiocompatible gellable material directly surrounding biologically activematerial. Although the actual dimensions of the invention microcapsulesare not critical, the term “microcapsules” includes capsules ofbiocompatible gellable material the largest dimensions of whichtypically falls in the range of about 1 μm up to about 1000 μm, with apreferable largest dimension falling in the range of about 100 μm up toabout 800 μm. Commonly, all dimensions of the microcapsule exceed 20 nm.Invention microcapsules can be produced in a variety of shapes, i.e., inthe shape of a cylinder (i.e., a geometrical solid generated by therevolution of a rectangle about one of its sides), a sphere (i.e., asolid geometrical figure generated by the revolution of a semicirclearound its diameter), a disc (i.e., a generally flat, circular form), aflat sheet (i.e., a generally flat polygonal form, preferably square orrectangular), a wafer (i.e., an irregular flat sheet), a dog-bone (i.e.,a shape that has a central stem and two ends which are larger indiameter than the central stem, such as a dumbbell), or the like.Invention microcapsules are generally formed so that the pore size of atleast the outer layer of the microcapsule is sufficiently large to allowunhindered diffusion of:

[0016] the biologically active material contained therein, or

[0017] the compound secreted by the biologically active materialcontained therein, or

[0018] the compound to be catalyzed and/or reacted by the biologicallyactive material contained therein.

[0019] Invention microcapsules are generally formed so that the poresize of at least the outer layer of the microcapsule is sufficientlysmall to block inward difflusion of molecules which are capable ofinitiating an immune response to the biologically active material (e.g.,IgG, complement proteins, and the like), at least when the microcapsuleis formed to not be contained within a larger macrocapsule.

[0020] Biologically active materials contemplated for containment and/ordelivery in accordance with the present invention include individualliving cells or groups of living cells (e.g., cell aggregates),biological materials (for diagnostic purposes, e.g., for in vivoevaluation of the effects of such biological materials on an organism,and conversely, the effects of the organism on such biologicalmaterials), pharmacologically active drugs, diagnostic agents, agents ofnutritional value, hemoglobin (to create artificial blood), and thelike.

[0021] As utilized herein, the term “living cells” includes any viablecellular material, regardless of the source thereof. Thus, virus cells,prokaryotic cells, eukaryotic cells, plant cells, and the like, arecontemplated. Specifically contemplated living cells include islets ofLangerhans (for the treatment of diabetes) (including individualpancreatic islet cells (e.g., α, β, and δ cells of pancreatic islets),tumor cells (for evaluation of chemotherapeutic agents), humanT-lymphoblastoid cells sensitive to the cytopathic effects of HIV,dopamine secreting cells (for the treatment of Parkinson's disease),nerve growth factor cells (for the treatment of Alzheimer's disease),hepatocytes (for treatment of liver dysfunction), adrenalin/angiotensinsecreting cells (for regulation of hypo/hypertension), parathyroid cells(for replacing thyroid function), norepinephrine/metencephalin secretingcells (for the control of pain), and the like. These living cells can beindividual cells, or aggregates of cells held together via intercellularadhesion mechanisms characteristic of the individual cells (e.g.,islets, and the like).

[0022] Examples of pharmacologically active agents include:

[0023] analgesics/antipyretics (e.g., aspirin, acetaminophen, ibuprofen,naproxen sodium, buprenorphine hydrochloride, propoxyphenehydrochloride, propoxyphene napsylate, meperidine hydrochloride,hydromorphone hydrochloride, morphine sulfate, oxycodone hydrochloride,codeine phosphate, dihydrocodeine bitartrate, pentazocine hydrochloride,hydrocodone bitartrate, levorphanol tartrate, diflunisal, trolaminesalicylate, nalbuphine hydrochloride, mefenamic acid, butorphanoltartrate, choline salicylate, butalbital, phenyltoloxamine citrate,diphenhydramine citrate, methotrimeprazine, cinnamedrine hydrochloride,meprobamate, and the like),

[0024] anesthetics (e.g., cyclopropane, enflurane, halothane,isoflurane, methoxyflurane, nitrous oxide, propofol, and the like),

[0025] antiasthmatics (e.g., Azelastine, Ketotifen, Traxanox, and thelike),

[0026] antibiotics (e.g., neomycin, streptomycin, chloramphenicol,cephalosporin, ampicillin, penicillin, tetracycline, and the like),

[0027] antidepressants (e.g., nefopam, oxypertine, doxepinhydrochloride, amoxapine, trazodone hydrochloride, amitriptylinehydrochloride, maprotiline hydrochloride, phenelzine sulfate,desipramine hydrochloride, nortriptyline hydrochloride, tranylcyprominesulfate, fluoxetine hydrochloride, doxepin hydrochloride, imipraminehydrochloride, imiprarnine pamoate, nortriptyline, arnitriptylinehydrochloride, isocarboxazid, desipramine hydrochloride, trimipraminemaleate, protriptyline hydrochloride, and the like),

[0028] antidiabetics (e.g., biguanides, hormones, sulfonylureaderivatives, and the like),

[0029] antifungal agents (e.g., griseofulvin, keloconazole, amphotericinB, Nystatin, candicidin, and the like),

[0030] antihypertensive agents (e.g., propanolol, propafenone,oxyprenolol, Nifedipine, reserpine, trimethaphan camsylate,phenoxybenzamine hydrochloride, pargyline hydrochloride, deserpidine,diazoxide, guanethidine monosulfate, minoxidil, rescinnamine, sodiumnitroprusside, rauwolfia serpentina, alseroxylon, phentolamine mesylate,reserpine, and the like),

[0031] anti-inflammatories (e.g., (non-steroidal) indomethacin,naproxen, ibuprofen, ramifenazone, piroxicam, (steroidal) cortisone,dexamethasone, fluazacort, hydrocortisone, prednisolone, prednisone, andthe like),

[0032] antineoplastics (e.g., adriamycin, cyclophosphamide, actinomycin,bleomycin, duanorubicin, doxorubicin, epirubicin, mitomycin,methotrexate, fluorouracil, carboplatin, carmustine (BCNU), methyl-CCNU,cisplatin, etoposide, interferons, camptothecin and derivatives thereof,phenesterine, paclitaxel and derivatives thereof, taxotere andderivatives thereof, vinblastine, vincristine, tamoxifen, etoposide,piposulfan, and the like),

[0033] antianxiety agents (e.g., lorazepam, buspirone hydrochloride,prazepam, chlordiazepoxide hydrochloride, oxazepam, clorazepatedipotassium, diazepam, hydroxyzine pamoate, hydroxyzine hydrochloride,alprazolam, droperidol, halazepam, chlormezanone, dantrolene, and thelike),

[0034] immunosuppressive agents (e.g., cyclosporine, azathioprine,mizoribine, FK506 (tacrolimus), and the like),

[0035] antimigraine agents (e.g., ergotamine tartrate, propanololhydrochloride, isometheptene mucate, dichloralphenazone, and the like),

[0036] sedatives/hypnotics (e.g., barbiturates (e.g., pentobarbital,pentobarbital sodium, secobarbital sodium, and the like),benzodiazapines (e.g., flurazepam hydrochloride, triazolam, tomazeparm,midazolam hydrochloride, and the like), and the like),

[0037] antianginal agents (e.g., beta-adrenergic blockers, calciumchannel blockers (e.g., nifedipine, diltiazem hydrochloride, and thelike), nitrates (e.g., nitroglycerin, isosorbide dinitrate,pentaerythritol tetranitrate, erythrityl tetranitrate, and the like),and the like),

[0038] antipsychotic agents (e.g., haloperidol, loxapine succinate,loxapine hydrochloride, thioridazine, thioridazine hydrochloride,thiothixene, fluphenazine hydrochloride, fluphenazine decanoate,fluphenazine enanthate, trifluoperazine hydrochloride, chlorpromazinehydrochloride, perphenazine, lithium citrate, prochlorperazine, and thelike),

[0039] antimanic agents (e.g., lithium carbonate and the like),

[0040] antiarrhythmics (e.g., bretylium tosylate, esmolol hydrochloride,verapamil hydrochloride, amiodarone, encainide hydrochloride, digoxin,digitoxin, mexiletine hydrochloride, disopyramide phosphate,procainamide hydrochloride, quinidine sulfate, quinidine gluconate,quinidine polygalacturonate, flecainide acetate, tocainidehydrochloride, lidocaine hydrochloride, and the like),

[0041] antiarthritic agents (e.g., phenylbutazone, sulindac,penicillamine, salsalate, piroxicam, azathioprine, indomethacin,meclofenamate sodium, gold sodium thiomalate, ketoprofen, auranofin,aurothioglucose, tolmetin sodium, and the like),

[0042] antigout agents (e.g., colchicine, allopurinol, and the like),

[0043] anticoagulants (e.g., heparin, heparin sodium, warfarin sodium,and the like),

[0044] thrombolytic agents (e.g., urokinase, streptokinase, altoplase,and the like),

[0045] antifibrinolytic agents (e.g., aminocaproic acid and the like),

[0046] hemorheologic agents (e.g., pentoxifylline and the like),

[0047] antiplatelet agents (e.g., aspirin, empirin, ascriptin, and thelike),

[0048] anticonvulsants (e.g., valproic acid, divalproate sodium,phenytoin, phenytoin sodium, clonazepam, primidone, phenobarbitol,phenobarbitol sodium, carbamazepine, amobarbital sodium, methsuximide,metharbital, mephobarbital, mephenytoin, phensuximide, paramethadione,ethotoin, phenacemide, secobarbitol sodium, clorazepate dipotassium,trimethadione, and the like),

[0049] antiparkinson agents (e.g., ethosuximide, and the like),

[0050] antihistamines/antipruritics (e.g., hydroxyzine hydrochloride,diphenhydramine hydrochloride, chlorpheniramine maleate, brompheniraminemaleate, cyproheptadine hydrochloride, terfenadine, clemastine fumarate,triprolidine hydrochloride, carbinoxamine maleate, diphenylpyralinehydrochloride, phenindamine tartrate, azatadine maleate, tripelennaminehydrochloride, dexchlorpheniramine maleate, methdilazine hydrochloride,trimprazine tartrate and the like),

[0051] agents useful for calcium regulation (e.g., calcitonin,parathyroid hormone, and the like),

[0052] antibacterial agents (e.g., amikacin sulfate, aztreonam,chloramphenicol, chloramphenicol palmitate, chloramnphenicol sodiumsuccinate, ciprofloxacin hydrochloride, clindamycin hydrochloride,clindamycin palmitate, clindamycin phosphate, metronidazole,metronidazole hydrochloride, gentamicin sulfate, lincomycinhydrochloride, tobramycin sulfate, vancomycin hydrochloride, polymyxin Bsulfate, colistimethate sodium, colistin sulfate, and the like),

[0053] antiviral agents (e.g., interferon gamma, zidovudine, amantadinehydrochloride, ribavirin, acyclovir, and the like),

[0054] antimicrobials (e.g., cephalosporins (e.g., cefazolin sodium,cephradine, cefaclor, cephapirin sodium, ceftizoxime sodium,cefoperazone sodium, cefotetan disodium, cefutoxime azotil, cefotaximesodium, cefadroxil monohydrate, ceftazidime, cephalexin, cephalothinsodium, cephalexin hydrochloride monohydrate, cefamandole nafate,cefoxitin sodium, cefonicid sodium, ceforanide, ceftriaxone sodium,ceftazidime, cefadroxil, cephradine, cefuroxime sodium, and the like),penicillins (e.g., ampicillin, amoxicillin, penicillin G benzathine,cyclacillin, ampicillin sodium, penicillin G potassium, penicillin Vpotassium, piperacillin sodium, oxacillin sodium, bacampicillinhydrochloride, cloxacillin sodium, ticarcillin disodium, azlocillinsodium, carbenicillin indanyl sodium, penicillin G potassium, penicillinG procaine, methicillin sodium, nafcillin sodium, and the like),erythromycins (e.g., erythromycin ethylsuccinate, erythromycin,erythromycin estolate, erythromycin lactobionate, erythromycin siearate,erythromycin ethylsuccinate, and the like), tetracyclines (e.g.,tetracycline hydrochloride, doxycycline hyclate, minocyclinehydrochloride, and the like), and the like),

[0055] anti-infectives (e.g., GM-CSF and the like),

[0056] bronchodialators (e.g., sympathomimetics (e.g., epinephrinehydrochloride, metaproterenol sulfate, terbutaline sulfate, isoetharine,isoetharine mesylate, isoetharine hydrochloride, albuterol sulfate,albuterol, bitolterol, mesylate isoproterenol hydrochloride, terbutalinesulfate, epinephrine bitartrate, metaproterenol sulfate, epinephrine,epinephrine bitartrate), anticholinergic agents (e.g., ipratropiumbromide), xanthines (e.g., aminophylline, dyphylline, metaproterenolsulfate, aminophylline), mast cell stabilizers (e.g., cromolyn sodium),inhalant corticosteroids (e.g., flurisolidebeclomethasone dipropionate,beclomethasone dipropionate monohydrate), salbutamol, beclomethasonedipropionate (BDP), ipratropium bromide, budesonide, ketotifen,salmeterol, xinafoate, terbutaline sulfate, triamcinolone, theophylline,nedocromil sodium, metaproterenol sulfate, albuterol, flunisolide, andthe like),

[0057] hormones (e.g., androgens (e.g., danazol, testosterone cypionate,fluoxymesterone, ethyltostosterone, testosterone enanihate,methyltestosterone, fluoxymesterone, testosterone cypionate), estrogens(e.g., estradiol, estropipate, conjugated estrogens), progestins (e.g.,methoxyprogesterone acetate, norethindrone acetate), corticosteroids(e.g., triamcinolone, betamethasone, betamethasone sodium phosphate,dexamethasone, dexamethasone sodium phosphate, dexamethasone acetate,prednisone, methylprednisolone acetate suspension, triamcinoloneacetonide, methylprednisolone, prednisolone sodium phosphatemethylprednisolone sodium succinate, hydrocortisone sodium succinate,methylprednisolone sodium succinate, triamcinolone hexacatonide,hydrocortisone, hydrocortisone cypionate, prednisolone, fluorocortisoneacetate, paramethasone acetate, prednisolone tebulate, prednisoloneacetate, prednisolone sodium phosphate, hydrocortisone sodium succinate,and the like), thyroid hormones (e.g., levothyroxine sodium and thelike), and the like,

[0058] hypoglycemic agents (e.g., human insulin, purified beef insulin,purified pork insulin, glyburide, chlorpropamide, glipizide,tolbutamide, tolazamide, and the like),

[0059] hypolipidemic agents (e.g., clofibrate, dextrothyroxine sodium,probucol, lovastatin, niacin, and the like),

[0060] proteins (e.g., DNase, alginase, superoxide dismutase, lipase,and the like),

[0061] nucleic acids (e.g., sense or anti-sense nucleic acids encodingany therapeutically useful protein, including any of the proteinsdescribed herein, and the like),

[0062] agents useful for erythropoiesis stimulation (e.g.,erythropoietin and the like),

[0063] antiulcer/antireflux agents (e.g., famotidine, cimetidine,ranitidine hydrochloride, and the like),

[0064] antinauseants/antiemetics (e.g., meclizine hydrochloride,nabilone, prochlorperazine, dimenhydrinate, promethazine hydrochloride,thiethylperazine, scopolamine, and the like),

[0065] oil-soluble vitamins (e.g., vitamins A, D, E, K, and the like),

[0066] as well as other drugs such as mitotane, visadine,halonitrosoureas, anthrocyclines, ellipticine, and the like, and thelike.

[0067] Examples of diagnostic agents contemplated for use in thepractice of the present invention include ultrasound contrast agents,radiocontrast agents (e.g., iodo-octanes, halocarbons, renografin, andthe like), magnetic contrast agents (e.g., fluorocarbons, lipid solubleparamagnetic compounds, and the like), as well as other diagnosticagents which cannot readily be delivered without some physical and/orchemical modification to accommodate the substantially water insolublenature thereof.

[0068] Examples of agents of nutritional value contemplated for use inthe practice of the present invention include amino acids, sugars,proteins, carbohydrates, fat-soluble vitamins (e.g., vitamins A, D, E,K, and the like) or fat, or combinations of any two or more thereof.

[0069] As utilized herein, the term “ionically crosslinkable” means theability of a biocompatible gellable material to form ionicallycrosslinked networks in the presence of multivalent cation(s) such ascalcium, zinc, barium, strontium, aluminum, iron, manganese, nickel,cobalt, copper, cadmium, lead, and the like, or mixtures of any two ormore thereof. This ability is due to the interaction of anions of thebiocompatible gellable material (e.g., carboxy groups on alginate) toionically bond with the multivalent cations. Preferred multivalentcations include calcium, barium, and strontium, with calcium beingpresently preferred for ionically crosslinking a biocompatible gellablematerial comprising alginate.

[0070] The characterization of an ionically crosslinkable biocompatiblegellable material (or any portion thereof) as being “not ionicallycrosslinked” indicates that an insufficient amount of multivalentcation(s) required to substantially crosslink the biocompatible gellablematerial is present in the biocompatible gellable material, eitherbecause an insufficient amount was always present or because an amountof such multivalent cation(s) was removed by subjecting thebiocompatible gellable material to conditions sufficient tosubstantially disrupt ionic crosslinking in the biocompatible gellablematerial.

[0071] Biocompatible gellable materials contemplated for use in thepractice of the present invention include ionically crosslinkablematerials, covalently crosslinkable materials, polyionicallycrosslinkable materials, and the like, and mixtures of any two or morethereof.

[0072] Ionically crosslinkable materials contemplated for use in thepractice of the present invention include anionic materials which areionically crosslinkable (e.g., alginates and other polysaccharides,chitosan, gellan gum, xanthan gum, hyaluronic acid, heparin, pectin,carrageenan, and the like), covalently crosslinkable derivativesthereof, and the like, and mixtures of any two or more thereof.Alginates contemplated for use in the present invention include highG-content alginate, high-M content alginate, sodium alginate, and thelike, and mixtures of any two or more thereof.

[0073] Capsule properties like mechanical strength, pore size, andbiocompatibility can be varied with the type and concentration of thealginate employed. For example, alginates with differing α-L-guluronicacid (G blocks) to β-D-mannuronic acid (M blocks) ratios are capable ofyielding capsules with significantly differing properties. G blocks havea higher multivalent cation binding capacity than M blocks. In addition,alginates having higher fractions of G blocks are more biocompatiblethan those containing a larger fraction of M blocks since high M blockalginates have been found to induce fibrotic overgrowth. Accordingly,capsules synthesized from alginates with high G/M ratios are generallystronger and more biocompatible than those capsules synthesized fromalginates with lower G/M ratios. Thus, the use in accordance with thepresent invention of alginates having at least 60% or greater G blocksis preferred, with alginates having at least 70% or greater G blocksbeing presently preferred.

[0074] As a further example, alginates with differing molecular weights(MW) or alginate concentrations are capable of yielding capsules withsignificantly differing properties relating to mechanical strength, poresize, and biocompatibility of the capsule. Thus, it is possible tofurther modify the end properties of the capsule by choosing alginatesof specific types.

[0075] Polyionically crosslinkable materials contemplated for use in thepractice of the present invention include mixtures of ionicallycrosslinkable materials and polycationic materials and the like.Polycationic materials contemplated for use in the present inventioninclude polyamnino acids (e.g., polyhistidine, polylysine, polyomithine,and the like), polymers containing primary amine groups, secondary aminegroups, tertiary amine groups, or pyridinyl nitrogen(s) (such aspolyethyleneimine, polyallylamine, polyetheramine, polyvinylpyridine,and the like), covalently crosslinkable derivatives thereof, and thelike. Polycationic material molecular weight can vary, depending on thedegree of permeability desired. Polycationic material molecular weightswill typically fall within a range of about 1,000 to about 100,000 orhigher, with a presently preferred molecular weight in the range ofabout 10,000 up to about 50,000. Presently, preferred polycationicmaterials for use in the practice of the present invention includepolylysine (i.e., poly-D-lysine (PDL), poly-DL-lysine, poly-L-lysine(PLL), poly-ε-CBZ-D-lysine, poly-ε-CBZ-DL-lysine, poly-ε-CBZ-L-lysine),polyornithine (i.e., poly-DL-ornithine, poly-L-ornithine, orpoly-δ-CBZ-DL-ornithine), and the like, and mixtures of any two or morethereof.

[0076] Covalently crosslinkable materials contemplated for use in thepractice of the present invention include covalently crosslinkablepolysaccharides (e.g., covalently crosslinkable alginates), covalentlycrosslinkable polyethylene glycols (i.e., covalently crosslinkablePEGs), covalently crosslinkable polycationic materials, covalentlycrosslinkable proteins, covalently crosslinkable peptides, othercovalently crosslinkable synthetic polymers, and the like, and mixturesof any two or more thereof.

[0077] Covalently crosslinkable alginates contemplated for use in thepractice of the present invention include alginates modified with asubstituent X which is capable of undergoing free radical polymerization(X is a moiety containing a carbon-carbon double bond or triple bondcapable of free radical polymerization; and X is linked covalently tothe alginate through linkages selected from ester, ether, thioether,disulfide, amide, imide, secondary amines, tertiary amines, directcarbon-carbon (C—C) linkages, sulfate esters, sulfonate esters,phosphate esters, urethanes, carbonates, and the like). Examples ofcovalently crosslinkable alginates include allyl and vinyl ethers ofalginate, acrylate and methacrylate esters of alginate, and the like.

[0078] Covalently crosslinkable PEGs contemplated for use in thepractice of the present invention include linear or branched chain PEGs(including STAR PEGs) modified with a substituent X which is capable ofundergoing free radical polymerization (as described above); wherein Xis linked covalently to the PEG through linkages selected from ester,ether, thioether, disulfide, amide, imide, secondary amines, tertiaryamines, direct carbon-carbon (C-C) linkages, sulfate esters, sulfonateesters, phosphate esters, urethanes, carbonates, and the like. Examplesof such covalently crosslinkable PEGs include vinyl and allyl ethers ofPEG; acrylate, diacrylate and methacrylate esters of PEG; and the like;and mixtures of any two or more thereof.

[0079] PEGs having a wide range of molecular weights can be employed inthe practice of the present invention. Thus, mixtures of differentmolecular weights for covalently crosslinkable PEGs contemplated for usein the practice of the present invention include PEGs having a MW in therange of about 200 up to about 1,000,000 (with PEGs having molecularweights in the range of about 500 up to about 100,000 being preferred,and PEGs having molecular weights in the range of about 1000 up to about50,000 being presently preferred). Such PEGs can be linear or branchedchain (including STAR PEGs). STAR PEGs are molecules having a centralcore (such as divinyl benzene) which is anionically polymerizable undercontrolled conditions to form living nuclei having a predeterminednumber of active sites. Ethylene oxide is added to the living nuclei andpolymerized to produce a known number of PEG “arms,” which are quenchedwith water when the desired molecular weight is achieved. Alternatively,the central core can be an ethoxylated oligomeric glycerol that is usedto initiate polymerization of ethylene oxide to produce a STAR PEG ofdesired molecular weight.

[0080] Covalently crosslinkable polycationic materials contemplated foruse in the practice of the present invention include polycationicmaterials modified with a substituent X which is capable of undergoingfree radical polymerization (as described above); wherein X is linkedcovalently to the polycationic material through linkages selected fromester, ether, thioether, disulfide, amide, imide, secondary amines,tertiary amines, direct carbon-carbon (C—C) linkages, sulfate esters,sulfonate esters, phosphate esters, urethanes, carbonates, and the like.Examples of covalently crosslinkable polycationic materials includeallyl and vinyl ethers of polycations, acrylate and methacrylate estersof polycations, and the like.

[0081] Free radical polymerization of the above-described covalentlycrosslinkable materials can be carried out in a variety of ways, forexample, initiated by irradiation with suitable wavelengthelectromagnetic radiation (e.g., visible or ultraviolet radiation) inthe presence of a suitable photoinitiator, and optionally, cocatalystand/or comonomer. Alternatively, free radical polymerization can beinitiated by thermal initiation by a suitable free radical catalyst.

[0082] A variety of free radical initiators, as readily recognized bythose of skill in the art, can be employed in the practice of thepresent invention. Thus, photoinitiators, thermal initiators, and thelike can be employed. For example, suitable WV initiators include2,2-dimethoxy-2-phenyl acetophenone and its water soluble derivatives,benzoin ethyl ether, 2,2-dimethyl phenoxyacetophenone, benzophenone andits water soluble derivatives, benzil and its water soluble derivatives,thioxanthone and its water soluble derivatives, and the like. Forvisible light polymerization, a system of dye (also known as initiatoror photosensitizer) and cocatalyst (also known as cosynergist,activator, initiating intermediate, quenching partner, or free radicalgenerator) are used. Examples of suitable dyes are ethyl eosin, eosin,eosin Y, erythrosin, riboflavin, fluorscein, rose bengal, methyleneblue, thionine, and the like; examples of suitable cocatalysts aretriethanolamine, arginine, methyl diethanolamine, triethylamine, and thelike.

[0083] A small amount of a comonomer can optionally be added to thecrosslinking reaction to increase the polymerization rates. Examples ofsuitable comonomers include vinyl pyrrolidinone, acrylamide,methacrylamide, acrylic acid, ethacrylic acid, sodium acrylate, sodiummethacrylate, hydroxyethyl acrylate, hydroxyethyl methacrylate (HEMA),ethylene glycol diacrylate, ethylene glycol dimethacrylate,pentaerythritol triacrylate, pentaerythritol trimethacrylate,trimethylol propane triacrylate, trimethylol propane trimethacrylate,tripropylene glycol diacrylate, tripropylene glycol dimethacrylate,glyceryl acrylate, glyceryl methacrylate, and the like.

[0084] Photoinitiators, cocatalysts, and comonomers are collectivelyreferred to as photocomponents, and comprise the active components ofthe photopolymerizing solution.

[0085] Varying the concentrations and proportions of these components inthe photopolymerizing solution can be used to yield capsules withdifferent mechanical strengths and differing permeabilities. Thesefactors influence the in vivo shelf life and performance of the capsuleafter transplantation into the body. In accordance with the presentinvention, varying the type and amount of the components in thephotopolymerizing solution provides a means of controlling capsuleproperties like mechanical strength, porosity or permeability, andbiocompatibility.

[0086] Thus, increasing the contact time of the capsule with thephotopolymerizing solution gives more time for the photocomponents todiffuse inwards into the capsule. Such enhanced penetration distance,upon photopolymerization, results in a capsule with a greater extent ofcovalent bonding therethrough. Hence, contact times of the capsule withthe photopolymerizing solution determine the depth ofphotopolymerization relative to the size of the capsule. A capsule thathas been uniformly photopolymerized throughout its volume would beexpected to result in a homogeneous alginate matrix held togethercoherently by a uniformly distributed network of covalent bonds, in theabsence of interspersed ionic bonds. Such a situation arises when thecontact time is sufficiently long that photocomponents are allowed todiffuse throughout the entire volume of the capsule. On the other hand,a capsule that has been photopolymerized only on the surface would beexpected to result in a composite capsule with a liquified (e.g.,substantially noncrosslinked) core surrounded by a photocrosslinkedmodified-alginate gel layer on the surface, in the absence ofinterspersed ionic bonds. Such a situation arises when the contact timeis so short that the photocomponents do not diffuse throughout theentire volume of the capsule, but are instead localized in a layer closeto the outer surface of the capsule. Upon photopolymerization, thecapsule therefore possesses an ionically crosslinked alginate coresurrounded by a skin of covalently crosslinked and ionically crosslinkedalginate. Subsequent disruption of ionic crosslinking via inventionmethods yields a composite capsule with a liquified core surrounded by acovalently crosslinked layer.

[0087] Capsules (e.g., microcapsules and macrocapsules) contemplated foruse in the practice of the present invention may be furthercharacterized as comprising an outer layer and a core. Typically, theouter layer of a capsule comprises that portion of the biocompatiblegellable material which is on the outer surface of the capsule, whilethe core of a capsule is that portion of the biocompatible gellablematerial which is not the outer layer.

[0088] Generally, the outer layer of a capsule has a thickness of atleast about {fraction (1/500)}^(th) of the largest dimension of thecapsule (e.g., 1 micron for 500 micron macrocapsule), with a thicknessof at least about {fraction (1/25)}^(th) to about {fraction (2/25)}^(th)of the largest dimension of the capsule (e.g., 20-40 microns for 500micron macrocapsule) being preferred, and a thickness of at least about{fraction (1/10)}^(th) of the largest dimension of the capsule (e.g., 50microns for 500 micron macrocapsule) being presently preferred. When theouter layer of a capsule has been covalently crosslinked and optionallypolyionically crosslinked, this enhanced thickness of the outer layerprovides enhanced immunogenic protection (e.g., enhanced prevention ofdirect exposure of any immunogenic agents at the capsule surface (e.g.,polycations, unencapsulated biologically active materials, and thelike)) and enhanced stability (e.g., stability to long-term exposure tophysiological conditions), when compared to prior art microcapsules.

[0089] The core of a capsule contemplated for use in the practice of thepresent invention can optionally be covalently crosslinked and/orionically crosslinked. Thus, in one aspect, invention microcapsules cancomprise a biocompatible gellable material whose core is ionicallycrosslinked. In a further aspect, invention microcapsules can comprise abiocompatible gellable material whose core is both covalentlycrosslinked and ionically crosslinked. In an additional aspect,invention microcapsules can comprise a biocompatible gellable materialwhose core is covalently crosslinked, but not ionically crosslinked. Inanother aspect, invention microcapsules can comprise a biocompatiblegellable material whose core is neither ionically crosslinked norcovalently crosslinked.

[0090] In accordance with the present invention, there are furtherprovided macrocapsules containing biologically active materials therein,optionally contained in at least one microcapsule therein. Inventionmacrocapsules comprise a first biocompatible gellable material which isionically crosslinkable and which contains the biologically activematerials (and optionally present microcapsules) therein. When themicrocapsules are not present within the macrocapsule, inventionmacrocapsules are further characterized in that at least the outer layerof the macrocapsule is covalently crosslinked and optionallypolyionically crosslinked, but not ionically crosslinked. When themicrocapsules are present within the macrocapsule, inventionmacrocapsules are further characterized in that at least the outer layerof the macrocapsule is covalently crosslinked, polyionicallycrosslinked, or both covalently crosslinked and polyionicallycrosslinked, but not ionically crosslinked, and each of themicrocapsules contained within invention macrocapsules comprises asecond biocompatible gellable material containing the biologicallyactive materials therein.

[0091] As utilized herein, “macrocapsule” includes capsules of gelmaterial surrounding biologically active material, optionally containedwithin at least one microcapsule. The term “macrocapsule” can include“macro-membranes,” “macrogels,” “gel entrapped microcapsules,” “lace,”“noodles,” “teabags,” “threads,” “worms,” and the like. Although theactual dimensions of the invention macrocapsules are not critical, theterm “macrocapsules” includes capsules of biocompatible gellablematerial the largest dimensions of which typically fall in the range ofabout 1000 μm up to about 50000 μm. Commonly, all dimensions of theinvention macrocapsules are greater than 20 nm. Invention macrocapsulescan be produced in a variety of shapes, i.e., in the shape of a cylinder(i.e., a geometrical solid generated by the revolution of a rectangleabout one of its sides), a sphere (i.e., a solid geometrical figuregenerated by the revolution of a semicircle around its diameter), a disc(i.e., a generally flat, circular form), a flat sheet (i.e., a generallyflat polygonal form, preferably square or rectangular), a wafer (i.e.,an irregular flat sheet), a dog-bone (i.e., a shape that has a centralstem and two ends which are larger in diameter than the central stem,such as a dumbbell), or the like. The macrocapsule is generally formedso that the pore size of at least the outer layer of the macrocapsule issufficiently large to allow unhindered diffusion of:

[0092] the biologically active material contained therein, or

[0093] the biologically active compound (e.g., insulin) secreted by thebiologically active material (e.g., pancreatic islet cells) containedtherein, or

[0094] the compound to be catalyzed and/or reacted by the biologicallyactive material contained therein,

[0095] while being sufficiently small to block inward diffusion ofmolecules which are capable of initiating an immune response to thebiologically active material (e.g., IgG, complement proteins, and thelike).

[0096] Like the core of the invention microcapsules, the core of theinvention macrocapsules can typically be covalently crosslinked and/orionically crosslinked. Thus, in one aspect, invention macrocapsulescomprise a core that is ionically crosslinked. In a further aspect,invention macrocapsules comprise a core that is both covalentlycrosslinked and ionically crosslinked. In an additional aspect,invention macrocapsules can comprise a core that is covalentlycrosslinked, but not ionically crosslinked. In another aspect, inventionmicrocapsules can comprise a core that is neither ionically crosslinkednor covalently crosslinked.

[0097] Similar to the outer layer of the invention microcapsules, theouter layer of the optional microcapsule(s) contemplated for use as partof the invention macrocapsules commonly is ionically crosslinked,covalently crosslinked, polyionically crosslinked, or any suitablecombination of any two or more thereof. Thus, in one aspect, inventionmacrocapsules can comprise at least one microcapsule, wherein at leastthe outer layer of the microcapsule(s) is covalently crosslinked. In anadditional aspect, invention macrocapsules can comprise at least onemicrocapsule, wherein the outer layer of the microcapsule(s) ispolyionically crosslinked. In another aspect, invention macrocapsulescan comprise at least one microcapsule, wherein at least the outer layerof the microcapsule(s) is ionically crosslinked.

[0098] Typically, the core of the optional microcapsule(s) contemplatedfor use as part of the invention macrocapsules is covalently crosslinkedand/or ionically crosslinked. Thus, in one aspect, the core of themicrocapsule(s) contemplated for use as part of the inventionmacrocapsules is ionically crosslinked. In a further aspect, the core ofthe microcapsule(s) contemplated for use as part of the inventionmacrocapsules is covalently crosslinked and ionically crosslinked. Inanother aspect, the core of the microcapsule(s) contemplated for use aspart of the invention macrocapsules is covalently crosslinked, but notionically crosslinked. In an additional aspect, the core of themicrocapsule(s) contemplated for use as part of the inventionmacrocapsules is neither ionically crosslinked nor covalentlycrosslinked.

[0099] Capsules (e.g., microcapsules and macrocapsules) can bemanufactured by various techniques known to those of skill in the art,including but not limited to interfacial polycondensation, emulsionpolymerization, simple and complex coacervation, thermal and ionicgelation, phase separation, electrostatic precipitation, solventevaporation, and mechanical agitation. The specific manufacturingtechnique employed is dictated by various factors, including thechemistry of the biocompatible gellable material (i.e., the capsuleshell material), the properties desired of the capsule manufacturedthereby, and the like.

[0100] Biocompatible gellable material (e.g., alginate)—containingmicrocapsules (and biocompatible gellable material—containingmacrocapsules which comprise microcapsules) are generally producedemploying a co-axial pneumatic nozzle. The biocompatible gellablematerial solution (which contains the encapsulant (e.g., thebiologically active material (for microcapsules and/or macrocapsules),or the microcapsules containing the biologically active material (formacrocapsules)) is extruded through the central bore, with air flowingaround the solution. The air pressure provides the force necessary tobreak up the extruded biocompatible gellable material solution intodroplets. In such a system, the droplet size can be altered by varyingthe ratio of the solution flow rate to the air flow rate. Increasing thelatter relative to the former yields smaller droplets.

[0101] Macrocapsules can also be synthesized by extruding thebiocompatible gellable material solution manually through a syringeattached with a needle. The droplets detach from the needle when thedrop size becomes big enough that the gravitational force tending todislodge the droplet from the needle exceeds the forces of surfacetension tending to keep the droplet attached to the needle. The size ofthe droplets can be controlled by choosing needles with an appropriategauge.

[0102] Once the droplets of biocompatible gellable material solutionhave been formed, they can be subjected to a variety of crosslinkingconditions.

[0103] In one variety of crosslinking conditions, the droplets ofbiocompatible gellable material solution are ionically crosslinked andcovalently crosslinked to form capsules, and then subjected toconditions sufficient to disrupt ionic crosslinking in at least theouter layer of the capsule. Under this aspect, the droplets are firstsubjected to conditions sufficient to ionically crosslink the ionicallycrosslinkable material solution. Typically, these conditions comprisecontacting the droplets with an ionic crosslinking medium containing atleast one multivalent cation(s) (e.g., calcium) to yield ionicallycrosslinked capsules (e.g., ionically crosslinked microcapsules andionically crosslinked macrocapsules). These ionically crosslinkedcapsules are subsequently (or, optionally, simultaneously) contactedwith the photopolymerizing solution for a predetermined amount of time.During this time, the components of the photopolymerizing solution (thatis, photoinitiators, cocatalysts, and/or comonomers) diffuse inwardsinto the ionically crosslinked capsule.

[0104] As readily recognized by those of skill in the art, thepredetermined time can be varied as a function of the size of thecapsule (i.e., smaller capsules have larger ratios of surface area tovolume, and thus require less time for equivalent difflusion ofphotocomponents), the concentration of the individual components in thephotopolymerizing solution and their concentrations relative to eachother (i.e., different concentrations yield different properties ofcapsule), and the like. In addition, the predetermined time can bealtered in order to vary the extent of the covalent crosslinkabilityrelative to the ionic crosslinkability of the capsule.

[0105] The ionically crosslinked capsule containing the photocomponentscan then optionally be subsequently transferred to another solutioncontaining a concentration of the multivalent cation(s) (e.g., Ca²⁺)which is sufficiently high to maintain an intact ionically crosslinkeddroplet, yet sufficiently low (and definitely lower than theconcentration of the ionically crosslink initiating first multivalentcation(s) solution) to prevent mineralization due to possible localsupersaturation of the multivalent cation (e.g., Ca²⁺) within thedroplet.

[0106] The ionically crosslinked capsules are then subsequently (or,optionally, simultaneously) subjected to covalent crosslinkingconditions (e.g., photopolymerization (such as under visible light fromhigh pressure 100 W mercury lamps (strong emission at wavelength ofabout 500nm to about 550 nm) or argon ion laser light (wavelength of 514nm at powers between about 10 mW to about 2 W))). The covalentcrosslinking time is generally rapid (on the order of milliseconds (forphotopolymerization via an argon ion laser) to seconds (forphotopolymerization via a mercury lamp)), and varies with theconcentrations of biocompatible gellable material, initiator,cocatalyst, and comonomers in the ionically crosslinked capsule.

[0107] The time interval between the ionic crosslinking and the covalentcrosslinking of the droplets can be varied. This time interval can varyfrom 0 seconds (e.g., simultaneously subjecting the droplets to ioniccrosslinking conditions and covalent crosslinking conditions) to about 5minutes. The shorter the time interval, the smaller the possibilityexists that the photocomponents will diffuse out of the droplets andweaken the covalent crosslinking process, and the greater theprobability that a stable covalently crosslinked capsule will be formed.This is especially applicable to smaller sized droplets (e.g.,microcapsules or smaller macrocapsules), as their larger ratios ofsurface area (e.g., diffusion surface) to volume increase the potentialfor loss of photocomponents, and decrease the probability that a stablecovalently crosslinked capsule will be formed.

[0108] Subsequent to ionic crosslinking and covalent crosslinking, thecapsules can optionally be rinsed thoroughly with saline in order toremove excess multivalent cation(s) (whose removal helps reduce thechance of biomineralization) and unreacted photocomponents (whoseremoval helps reduce potential toxicity effects of these photocomponentson the biologically active materials). The capsules can optionally beincubated at about 37° C. in a suitable culture medium.

[0109] Additional alternative treatments that can follow the covalentcrosslinking step include subjecting the capsule to conditionssufficient to disrupt ionic crosslinking in at least the outer layer ofthe capsule. This disruption of ionic crosslinking can promote migrationand aggregation of the biologically active material, as well astransport of the biologically active material or components secreted bythe biologically active material out of the capsule.

[0110] Thus, in accordance with the present invention, there areadditionally provided methods of making a microcapsule havingsubstantially no ionic crosslinking in at least the outer layer thereofand containing biologically active materials therein. Invention methodsfor making such microcapsules comprise:

[0111] subjecting a microcapsule, wherein at least the outer layerthereof is ionically crosslinked, and wherein at least the outer layerthereof is covalently crosslinked and optionally polyionicallycrosslinked, and which contains biologically active materials therein,to conditions sufficient to disrupt ionic crosslinking in at least theouter layer thereof,

[0112] thereby forming a microcapsule having substantially no ioniccrosslinking in at least the outer layer thereof.

[0113] In accordance with the present invention, there are furtherprovided methods of making a macrocapsule having substantially no ioniccrosslinking in at least the outer layer thereof and containingbiologically active materials therein, optionally contained within atleast one microcapsule. Invention methods for making such macrocapsulescomprise:

[0114] subjecting a macrocapsule, wherein at least the outer layerthereof is ionically crosslinked, and which contains biologically activematerials therein, to conditions sufficient to disrupt ioniccrosslinking in at least the outer layer thereof,

[0115] thereby forming a macrocapsule having substantially no ioniccrosslinking in at least the outer layer thereof. When microcapsules arenot present within the macrocapsule, the macrocapsule can be furthercharacterized in that at least the outer layer thereof is covalentlycrosslinked and optionally polyionically crosslinked. When microcapsulesare present within the macrocapsule, the macrocapsule can be furthercharacterized in that at least the outer layer thereof is covalentlycrosslinked, polyionically crosslinked, or both covalently crosslinkedand polyionically crosslinked.

[0116] Conditions sufficient to disrupt ionic crosslinking, either in atleast the outer layer or the core of a microcapsule, a macrocapsule ortheir constituent biocompatible gellable materials, include contactingthe relevant microcapsule, macrocapsule or their constituentbiocompatible gellable materials with a solution of sodium citrate,ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaaceticacid (DTPA), and other biocompatible chelators of multivalent cations,and the like, and mixtures of any two or more thereof, in aconcentration sufficient to chelate sufficient cations to substantiallydisrupt ionic crosslinking in the relevant microcapsule, macrocapsule ortheir constituent biocompatible gellable materials. In a preferredembodiment, conditions sufficient to disrupt ionic crosslinking in atleast the outer layer of a capsule or constituent biocompatible gellablematerial comprise contacting the capsule or constituent biocompatiblegellable material with a solution of sodium citrate having aconcentration in the range of about 15 mM to about 1M.

[0117] In an alternative embodiment of the invention, capsules which arecovalently crosslinked but not ionically crosslinked, are prepared bydirectly covalently crosslinking, without first ionically crosslinking,the droplets of biocompatible gellable solution formed as describedabove.

[0118] Thus, in accordance with this aspect of the present invention,there are provided alternative methods of making a microcapsulecontaining biologically active materials therein, wherein dropletscomprising a suspension of biologically active materials in a covalentlycrosslinkable carrier are simultaneously subjected to:

[0119] conditions sufficient to prevent substantial dissociationthereof, and

[0120] conditions sufficient to induce substantial covalent crosslinkingthereof,

[0121] thereby forming the microcapsule. Invention methods of makingmicrocapsules are optionally characterized, in one aspect, in that noionic crosslinking is required (although such ionic crosslinking may bepresent) to stabilize the droplet prior to covalent crosslinkingthereof, as the conditions sufficient to prevent substantialdissociation of the droplet perform the requisite stabilizing functionof ionic crosslinking.

[0122] As utilized herein, the term “covalently crosslinkable carrier”includes all covalently crosslinkable materials as described herein.

[0123] Conditions sufficient to prevent substantial dissociation of thedroplet include contacting the droplet with a medium which issubstantially immiscible with the droplet and which does notsubstantially inhibit the induction of covalent crosslinking. Mediawhich are substantially immiscible with the droplet include those mediawhich are capable of solvating less than 10% of the volume of thedroplet during the time period in which the droplet is in contact withthe media. Media which do not substantially inhibit the induction ofcovalent crosslinking include media which conduct sufficientelectromagnetic energy from an energy source to the droplet to enableinitiation of covalent crosslinking in the covalently crosslinkablecarrier, without destroying the functionality of the biologically activematerial.

[0124] Media which are substantially immiscible with the droplet andwhich do not substantially inhibit the induction of substantial covalentcrosslinking also comprise, for aqueous droplets comprisingbiocompatible gellable materials contemplated for use in accordance withthe present invention, soybean oil, coconut oil, safflower oil,sunflower oil, sesame oil, other vegetable oils, and the like. In apreferred embodiment, such a solution comprises soybean oil.

[0125] Conditions sufficient to induce substantial covalent crosslinkingof the droplet include irradiating the droplet with sufficient energy toinduce photocrosslinking of the covalently crosslinkable carrier. Thisenergy is generally in the form of electromagnetic radiation, such asvisible light, ultraviolet (UV) radiation, or lasers, although thisenergy can also include thermal energy. Two preferred embodiments ofsuch conditions include contacting the droplet with an argon ion laserat a wavelength of about 514 nm and at a power level in the range ofabout 10 mW to about 2 W for no more than about 50 milliseconds, andcontacting the droplet with a high pressure (e.g., about 100 W) mercurylamp for no more than about 5 minutes.

[0126] In accordance with another aspect of the present invention, thereare provided alternative methods of making a macrocapsule containingbiologically active materials therein, wherein droplets, comprising asuspension of the biologically active materials, optionally containedwithin at least one microcapsule, in a covalently crosslinkable carrier,are simultaneously subjected to:

[0127] conditions sufficient to prevent substantial dissociationthereof, and

[0128] conditions sufficient to induce substantial covalent crosslinkingthereof,

[0129] thereby forming the macrocapsule. Invention methods of makingmacrocapsules are optionally characterized, in one aspect, in that noionic crosslinking is required (although such ionic crosslinking may bepresent) to stabilize the droplet prior to covalent crosslinkingthereof, as the conditions sufficient to prevent substantialdissociation of the droplet perform the requisite stabilizing functionof ionic crosslinking.

[0130] Capsules which comprise individual cells capable of forming cellaggregates and which have been formed in accordance with the foregoinginvention methods can be further characterized in that they are capableof facilitating migration of the cells within the core of the capsules,and aggregation of the cells to form cell aggregates.

[0131] Thus, in accordance with the present invention, there areadditionally provided capsules (e.g., microcapsules and macrocapsules)containing at least one cell aggregate therein. Invention capsulescomprise an ionically crosslinkable biocompatible gellable material, andhave a core and an outer layer, wherein at least the outer layer of thecapsule is covalently crosslinked or polyionically crosslinked or bothcovalently crosslinked and polyionically crosslinked, but not ionicallycrosslinked, and wherein said at least one cell aggregate is containedwithin the core which is not ionically crosslinked.

[0132] When the capsule is a macrocapsule containing microcapsules,there are at least two possible embodiments of this aspect of theinvention. In a first embodiment, at least the core of themicrocapsule(s) of the macrocapsule is not ionically crosslinked, andthe cell aggregate(s) is formed and contained within the core of themicrocapsule(s) of the macrocapsule. In a second embodiment, at leastthe core of the macrocapsule is not tonically crosslinked, and the cellaggregate(s) is formed and contained within the core of themacrocapsule.

[0133] As utilized herein, “cell aggregate” includes an aggregation ofindividual living cells. Presently preferred cell aggregates includepseudo islets, which are aggregates of individual pancreatic islet cells(including α, β, or δ pancreatic islet cells). The cell aggregates areformed within the microenvironment created by a capsule. Such amicroenvironment as is present within the capsule provides a low-stressmedium for the aggregation of single cells into clumps of cells, or cellaggregates. The cell aggregates can, under at least some conditions,optionally be further characterized as exhibiting properties andfunctionality substantially identical to those of naturally occurringislets of corresponding cells in vivo. Accurate control over the averagenumber and size of cell aggregates encapsulated in each capsule can beachieved by controlling the number of cells present per capsule. Thus,the number of cells present per capsule could be varied by:

[0134] varying the culturing conditions for the unencapsulatedindividual art cells (i.e., varying the number of cell division cyclesexperienced by each unencapsulated individual cell), and/or

[0135] varying the encapsulating conditions for the unencapsulatedindividual cells (i.e., varying the number of individual cells permicrocapsule), and/or

[0136] varying the culturing conditions for the encapsulated individualcells (i.e., varying the number of cell division cycles experienced byeach encapsulated individual cell).

[0137] In accordance with the present invention, there are additionallyprovided methods of making a capsule containing at least one cellaggregate therein. Invention methods comprise subjecting a capsulecomprising an ionically crosslinked biocompatible gellable materialwherein at least the outer layer of the capsule is covalentlycrosslinked or polyionically crosslinked or both covalently crosslinkedand polyionically crosslinked, wherein said capsule encapsulates aplurality of individual cells, to conditions sufficient to disrupt ioniccrosslinking within the core of the capsule.

[0138] Invention methods of making a capsule containing at least onecell aggregate therein can be optionally characterized, in one aspect,in that the ionic interactions within the capsule are sufficientlyreduced to facilitate migration of the individual cells within thecapsule, thereby facilitating aggregation and formation of at least onecell aggregate within the capsule.

[0139] When the capsule is a macrocapsule containing microcapsules,there are at least two possible embodiments of this aspect of theinvention method. In a first embodiment, at least the core of themicrocapsule(s) of the macrocapsule is subjected to conditionssufficient to disrupt ionic crosslinking within the core of themicrocapsule(s) of the macrocapsule, thereby rendering the core notionically crosslinked, and the cell aggregate(s) is formed and containedwithin the core of the microcapsule(s) of the macrocapsule. In a secondembodiment, at least the core of the macrocapsule is subjected toconditions sufficient to disrupt ionic crosslinking within the core ofthe microcapsule(s) of the macrocapsule, thereby rendering the core notionically crosslinked, and the cell aggregate(s) is formed and containedwithin the core of the macrocapsule.

[0140] Proliferation of the individual cells within the capsule can bedesirable because the number, size and rate of formation of the cellaggregates can be directly proportional to the number of individualcells present within the capsule. Where proliferation of the individualpancreatic islet cells is desired, the invention methods of making acapsule containing at least one cell aggregate therein can optionallyfurther include the step of:

[0141] subjecting the capsule to conditions sufficient to promoteproliferation of the at least one individual cell.

[0142] This step can take place either before or after the step ofsubjecting the capsule to conditions sufficient to disrupt ioniccrosslinking within the core of the capsule, as described above.

[0143] Conditions sufficient to promote proliferation of the individualpancreatic cells include contacting the individual pancreatic cells witha suitable culture medium.

[0144] In accordance with the present invention, there are also provideddelivery systems for biologically active materials. Invention deliverysystems comprise the invention microcapsules and/or the inventionmacrocapsules. The biologically active materials contemplated for usewith the invention delivery systems include the biologically activematerials contemplated for use with the invention microcapsules and theinvention macrocapsules.

[0145] All references cited in this application are hereby incorporatedherein by reference in their entirety, including the entire contents ofU.S. patent application Ser. No. 09/076,339, Filed: May 11, 1998.

[0146] The invention will now be described in greater detail withreference to the following non-limiting examples. Those of ordinaryskill in the art, when guided by the teachings of the specification, maydiscover during the term of this patent other embodiments of thisinvention which fall within the scope of the appended claims.

EXAMPLE 1 Method of Testing Strength of Microcapsules

[0147] The effect of varying the concentrations of the componentscomprising the photopolymerizing solution (i.e., photoinitiators,cocatalysts, and comonomers) both individually and relative to eachother has been characterized based on evaluation of bead strength.Trends in bead strengths provide a window into understanding theefficiency of ionic and covalent crosslinking. In general, a higher beadstrength indicates a stronger or closely crosslinked matrix. Such amatrix is expected to have a smaller pore size due to the increaseddensity of covalent crosslinking between the polymer molecules. Based onsuch analysis, evaluating bead strengths provides an insight intomechanical properties, diffusional properties (porosity andpermeability), and in vivo end performance of such capsules used forxenotransplantation. Such experiments help construct an experimentaldatabase to intelligently manipulate capsule production conditions so asto yield capsules with desired end properties.

[0148] Modified alginate (acrylate derivatized alginate or AA) utilizedin this Example 1 was prepared by chemically modifying alginate by theincorporation of acrylate groups. The method for modification isincluded in U.S. Pat. No. 5,700,848, Issue Date: Dec. 23, 1997, theentire contents of which are hereby incorporated by reference herein.

[0149] Modified-alginate, ionically crosslinked beads (comprising 2% AA)of 700 μm average diameter were synthesized by the conventional coaxialpneumatic nozzle technique. Three photopolymerizing solutions were used:1×eosin (EE)/triethylamine (TEA)/vinyl pyrrolidinone (VP) (each at aconcentration of 0.025 g/L, 2.5 ml/L, and 5 ml/L respectively),2×EE/TEA/VP (each at a concentration of 0.05 g/L, 5 ml/L, and 10 ml/Lrespectively), and 1×eosin Y (EY)/TEA/VP (each at a concentration of0.025 g/L, 2.5 ml/L, and 5 ml/L respectively). The time of exposure ofthe beads to the photopolymerizing solution (also referred to as soakingtime) was systematically varied, while the photopolymerization time waskept constant at 5 minutes. The mechanical integrity of the covalentlycrosslinked beads thereby synthesized was tested by subsequent immersionin 1M sodium citrate, followed by immersion in deionized water. Strengthanalysis of the beads was performed using Texture Analyzer (Stable MicroSystems, UK). The results are presented in the following table: Soakingtime, Average strength of six microbeads, g min 1 X EE/TEA/VP 2 XEE/TEA/VP 1 X EY/TEA/VP 1 157.2 215.1 12.55 5 201.2 403.9 136.6

[0150] It is seen from the above table that as the concentrations of thephotocomponents are doubled, the bead strength increases significantly,when the rest of the processing conditions are identical. This isbecause higher concentrations of photocomponents can result in a greaterextent of covalent crosslinking contributing to the increased beadstrength. For the same photopolymerizing solution, increasing thesoaking time of the beads in the photopolymerizing solution results inbeads with increased strength upon polymerization. This is becauseincreased soaking time allows longer time for the photocomponents todiffuse in the bead resulting in a greater penetration distance.Subsequent photopolymerization results in a stronger bead due to agreater extent of covalent crosslinking. It is also seen from the abovetable that under identical experimental conditions and at identicalconcentrations, EE results in the formation of a stronger bead than EY.This can be attributed to the additional hydrophobic interactions EE iscapable of participating in as compared to EY, due to the morehydrophobic nature of EE compared to EY.

[0151] Additional experiments were done by systematically varying theconcentration of each of the components comprising the photopolymerizingsolution, while keeping the concentrations of the rest of the componentsidentical. In a certain range of concentrations it was found that eachof the photocomponents contribute to bead strength. That is, in acertain range of concentrations, increasing the concentration of thephotoinitiator, cocatalyst, and comonomer either individually or incombination relative to the other components results in beads withincreased strengths after photopolymerization.

[0152] Thus, varying the concentrations of components in thephotopolymerizing solution in addition to varying the soaking time ofthe beads in such solutions provides a convenient means of controllingcapsule properties like mechanical strength, porosity, and consequentlyin vivo end performance of the capsule after xenotransplantation.

EXAMPLE 2 Method of Forming Macrocapsules Having At Least Their OuterLayer Not Tonically Crosslinked

[0153] Modified alginate (acrylate derivatized alginate or AA) utilizedin this Example 2 was prepared in accordance with the method describedin Example 1.

[0154] Freshly harvested human islet cells were encapsulated in AAmacrocapsules by extruding a mixture of the AA (at a concentration of 2%in saline) and islet cells through a syringe attached with a 23 G or 21G needle into a Ca²⁺-rich solution (36 mM CaCl₂). The calcium ionicallycrosslinked the AA matrix, resulting in spherical ionically crosslinkedbeads approximately 2 mm in diameter. These beads were then immersed ina photopolymerizing solution consisting of eosin Y (0.025 g/l) (EY),triethyl amine (2.5 ml/l) (TEA), and vinyl pyrrolidinone (5 ml/l) (VP).The photocomponents were allowed to diffuse into the macrocapsules for 5min. The macrocapsules were subsequently transferred to a solution witha lower level of calcium (10 mM CaCl₂), and then immediatelyphotopolymerized using 100 W high pressure mercury lamps. Thephotopolymerization was carried out for 5 min. The resulting covalentlycrosslinked macrocapsules were washed thoroughly with saline to removeany unbound calcium or unreacted components of the photopolymerizingsolution.

[0155] The covalently crosslinked, ionically crosslinked macrocapsuleswere treated by immersion for 5 min in a sodium citrate solution (55mM). Such treatment yielded a macrocapsule solely crosslinked bycovalent linkages without the ionic linkages.

[0156] In vitro viability of the encapsulated human islets was examinedin this group of capsules by acridine orange/propidium iodide (AO/PI)staining. Function of the encapsulated islets was examined andquantified by static glucose stimulation (SGS). Unencapsulated (or free)islet viability and function was assessed similarly. Briefly, the SGStechnique involves stimulation of islets with a high level of glucoseand measurement of the secreted insulin (by RIA) in response to theglucose level. During SGS, either encapsulated or unencapsulated isletswere incubated in RPMI culture medium containing a basal level of 60 mg% glucose for 60 minutes, then transferred to a medium containing astimulatory level of 450 mg % glucose for 60 minutes, and returned tobasal medium (60 mg % glucose) for a further 60 minutes. The supernatantwas collected at the end of each 60 minute period. Insulin secretion wasassayed using RIA by measuring insulin concentration (μU/ml per isletequivalent count) in the supernatant.

[0157] An increase in the secreted insulin level above the basalsecretion during the stimulation phase, followed by a return in secretedinsulin to basal levels is a requisite for good islet function. Theviability of the islets in this group of capsules and in the free isletswas quite high (70-85%), indicating that the encapsulation environmentwas not toxic to the cells.

[0158] The encapsulated islets were also found to be functional in thegroup of capsules, yielding a SGS index of 5.14, as compared to an indexof 9.59 for free islets. In such tests, an index>3 is indicative ofhealthy islets. The encapsulated islets compared well to encapsulatedislets where there was no treatment with sodium citrate. The SGS indexfor these untreated, encapsulated islets was 6.01.

[0159] These tests show that the encapsulation system employed in thisexample (i.e., encapsulation followed by treatment with sodium citrate)is non-toxic to the cells, and the encapsulated islets remain healthyand retain normal function in such a microenvironment.

EXAMPLE 3 Method of Making Covalently Crosslinked Capsules Not RequiringPrior Ionic Crosslinking

[0160] Modified alginate (acrylate derivatized alginate or AA) utilizedin this Example 3 was prepared in accordance with the method describedin Example 1. In addition, modified PEG (PEG diacrylate or PEGDA) wasprepared by chemically modifying PEG by the incorporation of acrylategroups. The method for modification is included in U.S. patentapplication Ser. No. 09/076,339, Filed: May 11, 1998, the entirecontents of which are hereby incorporated by reference herein.

[0161] Photopolymerized AA and PEGDA coated capsules (e.g.,microcapsules and macrocapsules), which directly contained cells orcontained cells further encapsulated in alginate microcapsules, wereprepared in accordance with this Example 3.

[0162] The apparatus for synthesizing these capsules consisted of asystem of coaxial nozzles surrounded by an air jacket. The inner nozzlehad a 22G bore, and the outer nozzle had a 16G bore. The encapsulant (orthe biologic to be encapsulated) was to be extruded through the innernozzle, while the biocompatible gellable material was to besimultaneously extruded through the outer nozzle. Air/nitrogen was to bepumped through the outer jacket. The air flow rate was to be adjusted toyield capsules of differing sizes. For example, increasing the air flowrate relative to the liquid flow rate would result in synthesis ofsmaller capsules.

[0163] A suspension of cells (for microcapsule formation) or asuspension of the alginate microcapsules (for macrocapsule formation)was prepared. The alginate microcapsules with the cells encapsulated inthem were produced using the conventional coaxial pneumatic nozzlesystem.

[0164] AA and PEGDA were dissolved in a solution comprising deionizedwater, the photoinitiator (EY), cocatalyst (TEA), and comonomer (VP).The suspension (i.e., of cells, or of the alginate microcapsules) wasextruded through the inner nozzle, while the solution containing AA,PEGDA, and the photocomponents was simultaneously extruded through theouter nozzle. The extruded droplets were allowed to fall into soybeanoil. This resulted in a water-in-oil (w/o) emulsion, in which oilprevented the dissociation of the hydrophilic droplets. These dropletsin the w/o emulsion were simultaneously exposed to light from highpressure mercury lamps (100 watts). The photocomponents promotedcovalent crosslinking of the AA and PEGDA biocompatible gellablematerial in the presence of the mercury lamp light. This resulted in theformation of a capsule coated by a mixture of biocompatible materials(AA and PEGDA), in which each of the polymers is linked together (andpossibly to each other) by covalent crosslinking, and in which thecapsule is further characterized by an absence of ionic crosslinking.The core of such a capsule is either the cell suspension or a suspensionof alginate microspheres containing the cells.

[0165] The capsules can be isolated from the w/o emulsion by filtrationthrough a sieve with a suitable mesh rating, followed by repeatedwashings of the capsules with water. Alternatively, the capsules can berecovered by the addition of excess water in a separatory funnel,thereby allowing the hydrophilic capsules to migrate to the water phase.This can be done either in the presence or absence of a biocompatiblephase transfer agent. Repeated washings should be done to ensuresatisfactory removal of the oil phase.

EXAMPLE 4 Preparation of Microcapsules Containing Cell AggregatesTherein

[0166] Microcapsules, comprising unmodified alginate, a biocompatiblegellable material which is ionically crosslinkable, that encapsulatesindividual cells which are a coculture of α, β, and δ cells ofpancreatic islets, were synthesized by the conventional coaxialpneumatic nozzle technique. These microcapsules were then immersed in asolution of polylysine (PL), thereby resulting in a coating of PL aroundthe alginate capsules to form an outer layer of the biocompatiblegellable material which was polyionically crosslinked. The resultingmicrocapsules were unmodified alginate-polylysine (APL) microcapsules.After coating the microcapsules with PL, the core of these microcapsuleswas liquified by degelling them through immersion of the microcapsulesin sodium citrate (55 mM).

[0167] The microcapsules were then left standing. Upon standing, theindividual pancreatic islets cells within the microcapsule tended toaggregate in the microcapsule, resulting in the formation of cellaggregates.

[0168] This Example 4 demonstrates that cell aggregates can more easilyform in a capsule when the core of the capsule is not ionicallycrosslinked.

EXAMPLE 5 Preparation of Microcapsules For Aggregation of ProliferatedCells

[0169] Alginate-PLL microcapsules, ranging from 300-1000 μm in diameter,were synthesized by pneumatic coaxial extrusion in accordance with thetechniques described herein. The average size of the microcapsule in thecurrent application was 800 μm±70 μm.

[0170] The initial loading of human pancreatic single cells was in therange of 5×10⁶ to 15×10⁶ cells/ml of the alginate solution. In thisparticular application, the aforementioned cell loading translates toapproximately 1300-4000 single cells/microcapsule. Upon aggregation ofthe cells within the microcapsule after degelling the microcapsule viasodium citrate treatment and after contacting the microcapsule with asuitable culture medium, it was observed that a loading of 1-15pancreatic cell aggregates/microcapsule was achieved.

[0171] The “cell aggregates” formed as described above weremorphologically similar to that of native, freshly isolated humanislets, suggesting that the microcapsule indeed provided a low-stressenvironment for cell aggregation. These cell aggregates were both viableand functional as established through viability and function tests.

[0172] Cell viability was assessed by acridine orange/propidium iodide(AO/PI) staining, while function was assessed by Static GlucoseStimulation (SGS) tests. The cell aggregates had a viability≧75%(usually, 70-90%), indicating that the cell aggregates generated asdescribed above were healthy islets. SGS indicated a stimulation index(SI)≧2.0 (usually, 2.0≧SI≧40), suggesting that the aforementioned cellaggregates are capable of normal insulin secretion function. Successfulreversal of diabetes was achieved in STZ-induced diabetic rats aftertransplantation of the encapsulated cell aggregates into these rats.These tests indicate that the cell aggregates are healthy and viable,and are capable of both in vitro and in vivo function. Details of theAO/PI stain and SGS test referenced above are described in Example #2 ofU.S. patent application Ser. No. 09/076,339, Filed: May 11, 1998, theentire contents of which have already been incorporated herein byreference.

[0173] This Example 5 demonstrates that viable, functional cellaggregates can readily form in a capsule.

[0174] While the invention has been described in detail with referenceto certain preferred embodiments thereof, it will be understood thatmodifications and variations are within the spirit and scope of thatwhich is described and claimed.

That which is claimed is:
 1. A microcapsule containing biologicallyactive materials therein, the microcapsule comprising an ionicallycrosslinkable biocompatible gellable material, wherein at least theouter layer of said biocompatible gellable material is covalentlycrosslinked and optionally polyionically crosslinked, but not ionicallycrosslinked.
 2. The microcapsule according to claim 1, wherein the coreof said microcapsule is ionically crosslinked.
 3. The microcapsuleaccording to claim 2, wherein the core of said microcapsule iscovalently crosslinked.
 4. The microcapsule according to claim 1,wherein the core of said microcapsule is covalently crosslinked.
 5. Themicrocapsule according to claim 4, wherein the core of said microcapsuleis not ionically crosslinked.
 6. The microcapsule according to claim 1,wherein the core of said microcapsule is not ionically crosslinked.
 7. Amacrocapsule containing biologically active materials therein, saidmacrocapsule comprising a first biocompatible gellable material which isionically crosslinkable and which optionally contains at least onemicrocapsule therein, wherein, when at least one microcapsule ispresent, each microcapsule comprises a second biocompatible gellablematerial containing the biologically active materials therein and atleast the outer layer of said macrocapsule is covalently crosslinked orpolyionically crosslinked or both polyionically crosslinked andcovalently crosslinked, but not ionically crosslinked, and wherein, whenmicrocapsules are not present, at least the outer layer of said firstbiocompatible gellable material is covalently crosslinked and optionallypolyionically crosslinked, but not ionically crosslinked.
 8. Themacrocapsule according to claim 7, wherein the core of said macrocapsuleis ionically crosslinked.
 9. The macrocapsule according to claim 8,wherein the core of said macrocapsule is covalently crosslinked.
 10. Themacrocapsule according to claim 7, wherein the core of said macrocapsuleis covalently crosslinked.
 11. The macrocapsule according to claim 10,wherein the core of said macrocapsule is not ionically crosslinked. 12.The macrocapsule according to claim 7, wherein the core of saidmacrocapsule is not ionically crosslinked.
 13. The macrocapsuleaccording to claim 7, wherein at least the outer layer of each of saidmicrocapsules is covalently crosslinked.
 14. The macrocapsule accordingto claim 13, wherein the core of each of said microcapsules iscovalently crosslinked.
 15. The macrocapsule according to claim 8,wherein at least the outer layer of each of said microcapsules iscovalently crosslinked.
 16. The macrocapsule according to claim 15,wherein the core of each of said microcapsules is covalentlycrosslinked.
 17. The macrocapsule according to claim 9, wherein at leastthe outer layer of each of said microcapsules is covalently crosslinked.18. The macrocapsule according to claim 17, wherein the core of each ofsaid microcapsules is covalently crosslinked.
 19. The macrocapsuleaccording to claim 10, wherein at least the outer layer of each of saidmicrocapsules is covalently crosslinked.
 20. The macrocapsule accordingto claim 19, wherein the core of each of said microcapsules iscovalently crosslinked.
 21. The macrocapsule according to claim 11,wherein at least the outer layer of each of said microcapsules iscovalently crosslinked.
 22. The macrocapsule according to claim 21,wherein the core of each of said microcapsules is covalentlycrosslinked.
 23. The macrocapsule according to claim 12, wherein atleast the outer layer of each of said microcapsules is covalentlycrosslinked.
 24. The macrocapsule according to claim 23, wherein thecore of each of said microcapsules is covalently crosslinked.
 25. Adelivery system for biologically active materials comprising amicrocapsule according to claim 1, wherein said biologically activematerial is selected from the group consisting of living cells,biological materials, pharmacologically active drugs, and diagnosticagents.
 26. The delivery system according to claim 25, wherein saidbiologically active material comprises living cells.
 27. The deliverysystem according to claim 26, wherein said living cells are selectedfrom the group consisting of pancreatic islet cells, tumor cells, humanT-lymphoblastoid cells, islet of Langerhans cells, dopamine secretingcells, nerve growth factor cells, hepatocytes, adrenalin/angiotensinsecreting cells, parathyroid cells, and norepinephrine/metencephalinsecreting cells.
 28. The delivery system according to claim 25, whereinsaid biologically active material comprises biological materials. 29.The delivery system according to claim 25, wherein said biologicallyactive material comprises pharmacologically active drugs.
 30. Thedelivery system according to claim 25, wherein said biologically activematerial comprises diagnostic agents.
 31. The delivery system accordingto claim 25, wherein said biologically active material comprisespancreatic islet cells.
 32. A delivery system for biologically activematerials comprising a macrocapsule according to claim 7, wherein saidbiologically active material is selected from the group consisting ofliving cells, biological materials, pharmacologically active drugs, anddiagnostic agents.
 33. The delivery system according to claim 32,wherein said biologically active material comprises living cells. 34.The delivery system according to claim 33, wherein said living cells areselected from the group consisting of pancreatic islet cells, tumorcells, human T-lymphoblastoid cells, islet of Langerhans cells, dopaminesecreting cells, nerve growth factor cells, hepatocytes,adrenalin/angiotensin secreting cells, parathyroid cells, andnorepinephrine/metencephalin secreting cells.
 35. The delivery systemaccording to claim 32, wherein said biologically active materialcomprises biological materials.
 36. The delivery system according toclaim 32, wherein said biologically active material comprisespharmacologically active drugs.
 37. The delivery system according toclaim 32, wherein said biologically active material comprises diagnosticagents.
 38. The delivery system according to claim 32, wherein saidbiologically active material comprises pancreatic islet cells.
 39. Amethod of making a microcapsule containing biologically active materialstherein and having substantially no ionic crosslinking in at least theouter layer thereof, said method comprising:subjecting a microcapsulewhich contains biologically active materials therein, wherein at leastthe outer layer thereof is tonically crosslinked, and wherein at leastthe outer layer thereof is covalently crosslinked and optionallypolyionically crosslinked, to conditions sufficient to disrupt ioniccrosslinking in at least the outer layer thereof, thereby forming amicrocapsule having substantially no ionic crosslinking in at least theouter layer thereof.
 40. A method of making a macrocapsule containingbiologically active materials therein and having substantially no ioniccrosslinking in at least the outer layer thereof, said methodcomprising:subjecting a macrocapsule which contains biologically activematerials therein, optionally contained within at least one optionallypresent microcapsule, wherein at least the outer layer of themacrocapsule is ionically crosslinked, and wherein when microcapsulesare not present, at least the outer layer of the macrocapsule iscovalently crosslinked and optionally polyionically crosslinked, andwhen at least one microcapsule is present, at least the outer layer ofthe macrocapsule is covalently crosslinked or polyionically crosslinkedor both covalently crosslinked and polyionically crosslinked, toconditions sufficient to disrupt ionic crosslinking in at least theouter layer thereof, thereby forming a macrocapsule having substantiallyno ionic crosslinking in at least the outer layer thereof.
 41. A methodof making a microcapsule containing biologically active materialstherein, said method comprising simultaneously subjecting a dropletcomprising a suspension of biologically active materials in a covalentlycrosslinkable carrier to conditions sufficient to prevent substantialdissociation thereof, and subjecting the droplet to conditionssufficient to induce substantial covalent crosslinking thereof, therebyforming the microcapsule.
 42. The method of claim 41, wherein subjectingthe droplet to conditions sufficient to prevent substantial dissociationthereof comprises contacting the droplet with a medium which issubstantially immiscible with the droplet and which does notsubstantially inhibit the induction of covalent crosslinking.
 43. Themethod of claim 42, wherein the droplet is aqueous and the medium isselected from the group consisting of soybean oil, coconut oil,safflower oil, sunflower oil, and sesame oil.
 44. The method of claim42, wherein the droplet is aqueous and the medium comprises soybean oil.45. The method of claim 42, wherein subjecting the droplet to conditionssufficient to induce substantial covalent crosslinking comprisesirradiating the droplet with sufficient energy to inducephotocrosslinking of the covalently crosslinkable carrier.
 46. Themethod of claim 43, wherein subjecting the droplet to conditionssufficient to induce substantial covalent crosslinking comprisescontacting the droplet with light from high pressure mercury lamps for atime sufficient to induce photocrosslinking of the covalentlycrosslinkable carrier.
 47. A method of making a macrocapsule containingbiologically active materials therein, said method comprisingsimultaneously subjecting a droplet comprising a suspension of aplurality of microcapsules containing the biologically active materialsin a covalently crosslinkable carrier to conditions sufficient toprevent substantial dissociation thereof, and subjecting the droplet toconditions sufficient to induce substantial covalent crosslinkingthereof, thereby forcing the macrocapsule.
 48. The method of claim 47,wherein subjecting the droplet to conditions sufficient to preventsubstantial dissociation thereof comprises contacting the droplet with amedium which is substantially immiscible with the droplet and which doesnot substantially inhibit the induction of covalent crosslinking. 49.The method of claim 48, wherein the droplet is aqueous and the medium isselected from the group consisting of soybean oil, coconut oil,safflower oil, sunflower oil, and sesame oil.
 50. The method of claim48, wherein the droplet is aqueous and the medium comprises soybean oil.51. The method of claim 48, wherein subjecting the droplet to conditionssufficient to induce substantial covalent crosslinking comprisesirradiating the droplet with sufficient energy to inducephotocrosslinking of the covalently crosslinkable carrier.
 52. Themethod of claim 50, wherein subjecting the droplet to conditionssufficient to induce substantial covalent crosslinking comprisescontacting the droplet with light from high pressure mercury lamps for atime sufficient to induce photocrosslinking of the covalentlycrosslinkable carrier.
 53. A microcapsule containing at least one cellaggregate therein, said microcapsule having a core which is notionically crosslinked and an outer layer, wherein at least the outerlayer of the microcapsule is covalently crosslinked or polyionicallycrosslinked or both covalently crosslinked and polyionicallycrosslinked, but not ionically crosslinked, and wherein said at leastone cell aggregate is contained within the core.
 54. A macrocapsulecontaining at least one cell aggregate therein, said macrocapsulecomprising a first biocompatible gellable material which is covalentlycrosslinkable and which contains at least one microcapsule therein,wherein each microcapsule comprises a second biocompatible gellablematerial which is ionically crosslinkable, wherein at least the outerlayer of the macrocapsule is covalently crosslinked or polyionicallycrosslinked or both polyionically crosslinked and covalentlycrosslinked, wherein at least the outer layer of the microcapsule(s) iscovalently crosslinked or polyionically crosslinked or bothpolyionically crosslinked and covalently crosslinked, and wherein thecore of the microcapsule(s) is not ionically crosslinked and containssaid at least one cell aggregate.
 55. A macrocapsule containing at leastone cell aggregate therein, said macrocapsule comprising a firstbiocompatible gellable material which is ionically crosslinkable andcovalently crosslinkable and which optionally contains at least onemicrocapsule therein, wherein each microcapsule comprises a secondbiocompatible gellable material which is ionically crosslinkable,wherein at least the outer layer of the macrocapsule is covalentlycrosslinked or polyionically crosslinked or both polyionicallycrosslinked and covalently crosslinked, and wherein the core of themacrocapsule is not ionically crosslinked and contains said at least onecell aggregate.
 56. A method of making a microcapsule containing atleast one cell aggregate therein, said method comprising subjecting amicrocapsule comprising an ionically crosslinked biocompatible gellablematerial wherein at least the outer layer of the microcapsule iscovalently crosslinked or polyionically crosslinked or bothpolyionically crosslinked and covalently crosslinked, wherein saidmicrocapsule encapsulates at least one individual cell(s), to conditionssufficient to disrupt ionic crosslinking within the core of themicrocapsule, thereby facilitating proliferation and/or aggregation ofsaid individual cells to form at least one cell aggregate within themicrocapsule.
 57. The method of claim 56, said method further comprisingsubjecting the microcapsule to conditions sufficient to promoteproliferation of the at least one individual cell(s) after subjectingthe microcapsule to conditions sufficient to disrupt ionic crosslinkingwithin the core of the microcapsule.
 58. The method of claim 56, saidmethod further comprising subjecting the microcapsule to conditionssufficient to promote proliferation of the at least one individualcell(s) before subjecting the microcapsule to conditions sufficient todisrupt ionic crosslinking within the core of the microcapsule.
 59. Amethod of making a macrocapsule containing at least one cell aggregatetherein, said method comprising subjecting a macrocapsule comprising afirst biocompatible gellable material and at least one microcapsuletherein, wherein at least the outer layer of the macrocapsule iscovalently crosslinked or polyionically crosslinked or bothpolyionically crosslinked and covalently crosslinked, wherein each ofthe microcapsules comprises a second biocompatible gellable materialwhich is ionically crosslinked and which encapsulates at least oneindividual cell, wherein at least the outer layer of the at least onemicrocapsule is covalently crosslinked or polyionically crosslinked orboth polyionically crosslinked and covalently crosslinked, to conditionssufficient to disrupt ionic crosslinking within the core of the at leastone microcapsule, thereby facilitating proliferation and/or aggregationof said at least one individual cell to form at least one cell aggregatewithin the core of the microcapsule(s).
 60. The method of claim 59, saidmethod further comprising subjecting the macrocapsule to conditionssufficient to promote proliferation of said at least one individual cellafter subjecting the macrocapsule to conditions sufficient to disruptionic crosslinking within the core of the at least one microcapsule. 61.The method of claim 59, said method further comprising subjecting themacrocapsule to conditions sufficient to promote proliferation of saidat least one individual cell before subjecting the macrocapsule toconditions sufficient to disrupt ionic crosslinking within the core ofthe at least one microcapsule.
 62. A method of making a macrocapsulecontaining at least one cell aggregate therein, said method comprisingsubjecting a macrocapsule comprising a first biocompatible gellablematerial and at least one individual cell encapsulated therein,optionally contained within at least one optionally present microcapsuletherein, wherein at least the outer layer of the macrocapsule iscovalently crosslinked or polyionically crosslinked or bothpolyionically crosslinked and covalently crosslinked, wherein each ofthe microcapsules comprises a second biocompatible gellable materialwhich is ionically crosslinkable, to conditions sufficient to disruptionic crosslinking within microcapsule and at least the core of themacrocapsule, thereby facilitating proliferation and/or aggregation ofsaid individual pancreatic islet cells to form at least one cellaggregate within the core of the macrocapsule.
 63. The method of claim62, said method further comprising subjecting the macrocapsule toconditions sufficient to promote proliferation of the at least oneindividual cell after subjecting the macrocapsule to conditionssufficient to disrupt ionic crosslinking within the microcapsule and atleast the core of the macrocapsule.
 64. The method of claim 62, saidmethod further comprising subjecting the macrocapsule to conditionssufficient to promote proliferation of the at least one individual cellbefore subjecting the macrocapsule to conditions sufficient to disruptionic crosslinking within the microcapsule and at least the core of themacrocapsule.